Ornithine is a substance widely found in plants, animals, and microorganisms and is used as a precursor in the biosynthesis of arginine, proline, and polyamines. As a non-essential amino acid, ornithine is not found in proteins, but is present in peptide antibiotics such as tyrosidine and gramicidine. Ornithine plays an important role in the pathway for excretion of urea produced from amino acid or ammonia via the ornithine cycle in the in vivo metabolism of higher animals.
Ornithine helps to build muscles and to reduce body fat, and thus is used as a nutritional supplement. Ornithine-alpha ketoglutarate (OKG) containing ornithine and alpha ketoglutaric acid at a ration of 2:1 is used as an immune enhancer. Ornithine is also used a drugs for improving liver cirrhosis and liver function disorders, because it helps to remove harmful ammonia from the liver. The known methods of producing ornithine are treatment of milk casein with digestive enzymes and use of transformed E. coli or industrial microorganisms belonging to Corynebacterium sp., which are widely used in the production of amino acids, nucleic acids, enzymes and antibiotic-like substances.
In the microorganisms belonging to Corynebacterium sp., L-arginine is synthesized from glutamate by an enzyme expressed from a gene on the arginine operon in the form of argCJBDFRGH. The arginine operon genes, which play the most important role in arginine biosynthesis, synthesize arginine using intracellular glutamate (L-glutamate) as a substrate, and ornithine is produced as an intermediate during the syntheses of arginine. Specifically, as in FIG. 2 schematically illustrating a synthetic pathway of arginine from glutamate in a microorganism belonging to Corynebacterium sp., it is known that argJ encodes an enzyme converting glutamate to N-acetyl glutamate, argB encodes an enzyme converting N-acetyl glutamate to N-acetylglutamyl phosphate, argC encodes an enzyme converting N-acetylglutamyl phosphate to N-acetyl glutamate semialdehyde, argD encodes an enzyme converting N-acetyl glutamate semialdehyde to N-acetyl ornithine, argJ encodes an enzyme converting N-acetyl ornithine to ornithine, argF encodes an enzyme converting ornithine to citrulline, argG encodes an enzyme converting citrulline to argininosuccinate, and argH encodes an enzyme converting argininosuccinate to arginine in the arginine synthetic pathway, and the ornithine synthetic pathway is included in the arginine synthetic pathway.
The known arginine-producing strains have been developed by introducing a mutation into the arginine operon or by promoter mutation to increase the expression levels of the enzymes involved in the arginine biosynthesis. Of them, argR controlling and suppressing the arginine operon expression and argB inhibited by arginine level have been widely studied as targets for increasing arginine production (Korean Patent Publication No. 2010-0060909)
To improve ornithine productivity, it is known that ornithine production is increased by the action of ornithine cyclodeaminase (ocd) by culturing the Corynebacterium microorganism in a medium supplemented with proline, or by modifying impellers and culturing conditions during the culture of the microorganism. Also, when a transformed E. coli is used, ornithine productivity is improved by culturing argF and argR-deleted strains in a medium supplemented with glutamate, or by using a transformed strain with the deletion of proB gene encoding γ-glutamulkinase involved in the first step of the synthetic pathway of proline from glutamate rather that the synthetic pathway of ornithine form glutamate.
Furthermore, Corynebacterium glutamicum have been consistently studied for the high-yield production of glutamate as an ornithine precursor. Glutamate excretion from Corynebacterium glutamicum is known to be increased by biotin limitation of treatment with penicillin G or a fatty acid ester surfactant. Since these treatments are correlated with damage in the cell wall, it had previously been thought that glutamate leaks passively through the damaged cell wall.
NCgl1221 protein derived from the wild type Corynebacterium glutamicum (Cgl 13032) facilities betaine efflux, and its amino acid sequence is similar to that of the E. coli mechanosensitive channel protein, yggB (Korean Patent Publication No. 2010-0017581).